Comparative Assessment of Substrates and Activity Based...
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Comparative Assessment of Substrates and ActivityBased Probes as Tools for Non-Invasive Optical Imagingof Cysteine Protease ActivityGalia Blum1¤, Robby M. Weimer2, Laura E. Edgington1, Walter Adams1, Matthew Bogyo1,3*
1 Department of Pathology, Stanford University School of Medicine, Stanford, California, United States of America, 2 Department of Biomedical Imaging, Genentech Inc.,
South San Francisco, California, United States of America, 3 Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California,
United States of America
Abstract
Recent advances in the field of non-invasive optical imaging have included the development of contrast agents that reporton the activity of enzymatic targets associated with disease pathology. In particular, proteases have proven to be idealtargets for development of optical sensors for cancer. Recently developed contrast agents for protease activity include bothsmall peptides and large polymer-based quenched fluorescent substrates as well as fluorescently labeled activity basedprobes (ABPs). While substrates produce a fluorescent signal as a result of processing by a protease, ABPs are retained at thesite of proteolysis due to formation of a permanent covalent bond with the active site catalytic residue. Both methods havepotential advantages and disadvantages yet a careful comparison of substrates and ABPs has not been performed. Here wepresent the results of a direct comparison of commercially available protease substrates with several recently describedfluorescent ABPs in a mouse model of cancer. The results demonstrate that fluorescent ABPs show more rapid and selectiveuptake into tumors as well as overall brighter signals compared to substrate probes. These data suggest that the lack ofsignal amplification for an ABP is offset by the increased kinetics of tissue uptake and prolonged retention of the probesonce bound to a protease target. Furthermore, fluorescent ABPs can be used as imaging reagents with similar or betterresults as the commercially available protease substrates.
Citation: Blum G, Weimer RM, Edgington LE, Adams W, Bogyo M (2009) Comparative Assessment of Substrates and Activity Based Probes as Tools for Non-Invasive Optical Imaging of Cysteine Protease Activity. PLoS ONE 4(7): e6374. doi:10.1371/journal.pone.0006374
Editor: Jun Liu, Johns Hopkins School of Medicine, United States of America
Received May 16, 2009; Accepted June 25, 2009; Published July 28, 2009
Copyright: � 2009 Blum et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Institutes of Health National Technology Center for Networks and Pathways grant U54 RR020843, and NIH grantR01 EB005011 and a Susan G. Komen post-doctoral fellowship (to G.B.). The funders had no role in study design, data collection and analysis, decision to publish,or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected]
¤ Current address: School of Pharmacy, Faculty of Medicine, The Hebrew University, Jerusalem, Israel
Introduction
The past decade has seen a dramatic increase in the number of
new technologies that are available for applications in molecular
imaging and disease monitoring. The field of optical fluorescence
imaging has begun to show promise as a method that may soon
have significant clinical value. At the heart of all new imaging
methods is the need for contrast agents that provide a more precise
picture of distinct molecular events as they happen in vivo. Some of
the most recently developed classes of contrast agents are so-called
‘‘smart probes’’ that produce signal in response to a specific
enzyme mediated reaction. Because increased protease activity has
been shown to be associated with the pathogenesis of a number of
human diseases including cancer, atherosclerosis and neurode-
generative diseases, significant efforts have been made to develop
molecular sensors of protease activity. A large number of reagents
have been built around reporter substrates that, when cleaved by a
protease, produce a fluorescent signal (for review see[1,2]). These
reagents include large, polymer-based quenched fluorescent
substrates that are cleaved in multiple locations to produce
fluorescent products (Fig. 1A). As an alternative to substrates,fluorescent activity based probes have also been reported [3,4].
Unlike substrates, these reagents label target proteases through the
formation of a covalent bond with the active site cysteine (Fig. 1B).Because substrates and ABPs have highly distinct mechanisms of
action, a direct comparison of these two classes of agents in a
relevant biological system would be valuable for understanding the
strengths and weaknesses of each method.
Protease activity has historically been biochemically dissected
using protein and peptide substrates. One of the most common
tools used to monitor protease activity is a fluorogenic peptide
substrate. Various classes of fluorescent substrates have been
described, including those with reporters that change fluorescent
properties upon peptide bond hydrolysis, substrates containing
quencher or FRET reporter pairs that become separated by
protease cleavage of the peptide backbone and substrates that are
retained within cells when cleaved by a protease [5,6,7,8]. These
substrate types of have been designed using short peptide
sequences [5,9] or using fluorophores or fluorescent peptide
sequences tethered to a large polymer or dendramer backbone
[7,8]. Polymer-based substrates are internally quenched by the
high density of fluorophores loaded onto the backbone structure
and are designed to produce fluorescent products upon protease
processing. One such class of polymer-based reagents, ProSenseH
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(VisEn medical), is commercially available and an analog of this
probe family is currently moving towards early stage human
clinical trails (VisEn Medical Press Release June 16, 2008).
For all classes of substrates, selectivity for a given protease target
is controlled by the peptide recognition sequence. While some
proteases show a high degree of selectivity for a given sequence
(i.e. caspases which require a P1 aspartic acid) and can be targeted
with relatively selective substrates, others show high promiscuity
and are able to cleave a wide variety of peptide sequences.
Unfortunately, with more than 500 proteases in the human
genome [10], it remains extremely difficult to control selectivity of
substrate cleavage in vivo. In addition, the overall pharmacody-
namic properties of the substrate ultimately dictate which
proteases come into contact with the reporter and to what extent
a substrate will accumulate in a given tissue or organ. Regardless
of the drawbacks of selectivity, one of the major benefits of using a
substrate as a reporter is that a single active protease can process
many substrates, thus leading to signal amplification over time.
This amplification has been proposed to be a highly beneficial
property of substrate based imaging agents yet, the real
contribution of signal amplification has not been evaluated.
As an alternative to substrate based imaging agents, activity
based probes have recently been shown to have value for non-
invasive optical imaging applications [4]. One of the major
advantages of ABPs is that they covalently bind to a target
protease, allowing direct biochemical analysis of targets after in vivo
imaging has been performed. Furthermore, the selectivity of an
ABP can be controlled both by the peptide selectivity sequence
and the type of reactive functional group or ‘‘warhead’’ used on
the probe. Thus, it is possible to generate probes that have
exceedingly high selectivity for a small number of related
proteases, as has been demonstrated for the caspases and cysteine
cathepsins [11,12,13,14]. In addition, since ABPs tend to be small
molecules with relatively short half-lives in vivo they have the
potential to circulate quickly and be rapidly cleared resulting in the
production of high contrast images. However, since ABPs are also
inhibitors of their target enzymes, they bind only a single target
protease molecule and fluorescent signals are not amplified by
multiple processing events. Therefore, a direct comparison of
substrates and ABPs should provide information regarding the
value of signal amplification for optical imaging contrast reagents.
For this reason, we decided to perform a direct comparison of a
series of substrates and ABPs that have been independently
validated as imaging agents for the cysteine cathepsins. Specifi-
cally, we compared the commercially available ProSense polymer-
based substrates to the previously described fluorescently
quenched and non-quenched probes GB123, GB138 and
GB137 in a mouse model of cancer. For this study we co-injected
substrates and probes carrying non-overlapping fluorescent
reporters into the same animal and then compared fluorescent
images over time. In addition we evaluated probe uptake as well as
overall tissue distribution in vivo. To rule out the possibility of
inhibition of substrate signal by co-injection of the probes, we also
performed the comparison in separate animals with similar
outcome. Overall, our results indicate that the large polymer-
based substrates show slow uptake into tumors, have relatively
high background in organs such as liver and spleen and produce
overall weaker signals compared to the fluorescent ABPs. Based on
these findings we conclude that slow uptake and rapid diffusion of
the fluorescent products of substrates prevent signal amplification
from dramatically enhancing imaging signals and therefore ABPs
can be used with similar or better results.
Results
In order to directly compare substrate and ABP imaging agents,
we evaluated both classes of reagents in a simple xenograft mouse
model of cancer. For ABPs, we chose to use several recently
reported probes that target the cysteine cathepsins [3,4] (Fig. 1C).
Figure 1. Fluorescent protease probes for non-invasive imag-ing. (A) Schematic diagram illustrating activation of the ProSenseimaging probes by a target protease. The main backbone is made up ofPEGylated poly-lysine modified with fluorophores that are quenched byclose proximity (gray stars). Upon cleavage by a protease at free lysineresidues, smaller fragments containing the unquenched fluorophore(blue stars) are released. (B) Schematic of fluorescent activity basedprobes labeling a target protease. In both examples, the probecovalently binds in the active site of the protease target forming apermanent bond between the probe and the protease. In the topscheme, the probe contains a fluorescent reporter (blue star) that emitsfluorescence even in the absence of protease. In the bottom scheme,the fluorophore (gray star) on the ABP is quenched by proximity to aquenching group (black circle) that is lost upon covalent modificationof the target protease. (C) Structures of the ABPs used in this study. Theprobes GB123 and GB138 do not contain a quenching group and aretherefore always fluorescent. GB137 contains a Cy5 fluorophore that isquenched by proximity to the QSY quenching group.doi:10.1371/journal.pone.0006374.g001
Protease Substrates vs. ABPs
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These include the fluorescently quenched probe GB137 and the
non-quenched probes GB123 and GB138. All of these reagents
have the same general scaffold and reactive acyloxymethyl ketone
(AOMK) warhead group. The primary difference is the presence
of a QSY21 quenching group on the acyloxy leaving group of
GB137 and the use of an IR800 fluorophore in place of Cy5 on
GB138. As an initial comparison we chose to evaluate the
commercially available ProSenseH (VisEn Medical) substratesbecause they also have been shown to be activated by cathepsins.
These reagents consist of a pegylated poly-lysine backbone
derivatized with fluorophores. The high density of fluorophores
results in a polymer that is non-fluorescent due to self-quenching.
Upon cleavage of the poly-lysine backbone by a protease,
fluorescent fragments are released (Fig. 1A). Although thesereagents are effectively processed by multiple cathepsins, recent
data suggest that they can also be processed by other classes of
proteases (VisEn Medical website). Since substrates and ABPs use
fluorophores that emit light in the near infrared region, it is
possible to monitor their activation using whole body, non-invasive
imaging methods.
We initially evaluated the ProSense750 probe and GB137 in nude
mice bearing human MDA-MB 231 MFP tumors (Fig. 2). Since
Figure 2. Comparison of GB137 and ProSense750 using non-invasive optical imaging methods. (A) Images of GB137 and ProSense 750fluorescence in mice bearing MDA-MB 231 MFP tumors. Fluorescent images of live mice were taken at various time points after injection using anFMT1 imaging system. Representative pictures of the same mouse scanned over time in the 680 nm channel (GB137) and 750 nm channel(Prosense750) are shown. Fluorescent tomography scans (yellow box) are overlaid on an epifluorescent image. The colorometric scale bars indicatethe amount of fluorescence in the tomography box. (B) Quantification of total fluorescence in tumors. Fluorescence in tumors was measured in theindicated area (white box) and normalized to tumor weight. Mean fluorescence and standard error normalized to tumor weight for each channel isplotted relative to time after probe injection. Insert shows tumor fluorescence (in pmol) normalized to tumor weight at the 8 hour time point forGB137 and ProSense750 treated mice. GB137 fluorescence was corrected to account for the non-optimal filter set of the FMT1 (see methods and Fig.S1).doi:10.1371/journal.pone.0006374.g002
Protease Substrates vs. ABPs
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GB137 emits 666 nM light while ProSense750 emits 750 nM light,
we could inject both probes into the same animal and monitor each
probe individually using different filter sets. Probes were co-injected
via tail vein and overall fluorescence was monitored using
fluorescence tomography (FMT1, VisEn Medical) at various time
points (Fig. 2A). By using the FMT imaging system we were able tovisualize probe distribution in tumors as well as surrounding normal
tissues throughout the entire depth of the animal and obtain
quantitative readouts of total fluorescence in a given region of
interest. Images obtained at early time points after probe injection
(i.e. 40 min) show rapid and specific activation of the GB137 probe
with no signals observed for ProSense750. The ProSense750 probe
only showed measurable activation at the 8 hr time point, in
agreement with protocols for these agents in which imaging is
performed 24 hrs after probe injection[8]. In addition, we quantified
tumor fluorescence and corrected the signal intensity based on the
fluorophore used. Since the Cy5 and IR800 dyes used on the ABPs
do not fluoresce at a wavelength that is optimally measured using the
standard FMT filter set, we measured the total fluorescence intensity
of a probe standard to determine a scale factor that could be used to
accurately quantify the fluorescent signal for the ABPs (Fig. S1). Theresulting analysis confirmed that GB137 fluorescence was brighter
than the ProSense750 signal at the 8 hour time point (Fig. 2B).
We next compared the non-quenched ABP GB123 to the
ProSense750 substrate in the same xenograft tumor model
(Fig. 3A). Since GB123 is not quenched, it showed the expectedhigh background signals at the early time points as previously
reported [4]. However, we observed specific accumulation of the
probe in tumors at the 12 hour and 24 hour time points.
ProSense750 again showed specific tumor labeling after 8 hours
and had the brightest signal 24 hours after probe injection.
Quantification of total tumor fluorescence at the 24 hour time
point indicated an overall 10–12 fold brighter probe signal for the
ABP compared to the substrate (Fig. 3B).
Figure 3. Comparison of GB123 and ProSense750 using non-invasive optical imaging methods. (A) Images of GB123 and ProSense 750fluorescence in mice bearing MDA-MB 231 MFP tumors. Fluorescent images of live mice were taken at various time points after injection using anFMT1 imaging system. Representative pictures of the same mouse scanned over time in the 680 nm channel (GB123) and 750 nm channel(Prosense750) are shown. Fluorescent tomography scans (yellow box) are overlaid on an epifluoerscent image. The colorimetric scale bars indicatethe amount of fluorescence in the tomography box. (B) Quantification of total fluorescence in tumors. Fluorescence in tumors was measured in theindicated area (white box) and normalized to tumor weight. Mean fluorescence normalized to tumor weight and standard error for each channelwere plotted relative to time after probe injection. Insert shows the tumor fluorescence (in pmol) normalized to weight at the 24 hour time point ofGB123 and ProSense750 treated mice. GB123 fluorescence was corrected to account for the non-optimal filter set of the FMT1 (see methods and Fig.S1).doi:10.1371/journal.pone.0006374.g003
Protease Substrates vs. ABPs
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To determine if the fluorescent tag had a direct effect on probe
uptake and overall signal strength we co-injected tumor-bearing
mice with the IR800 labeled ABP GB138 and the ProSense680
probe (Fig. 4A). Since the GB138 probe is not quenched, itshowed high background labeling due to free probe circulation,
however tumor specific labeling was observed by 12 hours after
probe injection. Like GB123, GB138 showed good signal
accumulation in the tumor with high tumor to background ratios
at the late time points. In addition, the GB138 probe produced a
signal in tumors that was more than 3 times brighter than the
ProSense680 substrate at the 24 hour time point (Fig. 4B). Sincethe ABPs also act as covalent inhibitors, we wanted to make sure
that the ProSense signals were not reduced as the result of target
inhibition by the ABP. Therefore, we monitored the levels of
active cathepsin B in tumors from probe and control treated
animals. Analysis of tumor tissues from mice treated with GB138
at the same dose used for imaging studies confirmed that the
fluorescent probe efficiently labeled active cathepsin B and L.
However, tissues from both probe and control treated animals
showed similar levels of residual active cathepsin B, as measured
by ex vivo labeling of tissue extracts using a radiolabeled probe
(Fig. 4C). These data suggest that, at the doses used for imaging,GB138 only labels a sub-population of active cathepsins, leaving
the majority of the active enzyme intact.
To further confirm that co-injection of the ABP probes did not
artificially alter the ability of the ProSense probes to function, we
performed imaging studies in which mice were separately injected
with each of the probes. For these studies we compared all
substrates and ABPs in mice carrying tumors derived from the Ras
transformed mouse myoblast cell line C2C12 (Fig. 5). We havepreviously shown that these tumors grow faster and generally have
higher cathepsin activity than the MDA-MB-231 cells [4]. In
addition we performed imaging using the newly released
FMT2500, which allows tomographic imaging of animals without
the need for a density matching fluid. Direct comparison of probe
labeling in this model confirmed our earlier results. Specifically, we
found that only the quenched ABP GB137 provided specific probe
signal in tumors at the early time points. Again, the non-quenched
ABPs and the ProSense680 probe showed good contrast in tumors
at the 5 and 24 hr time points. Quantification of the tumor
fluorescence normalized to tumor weight indicated that the Cy5
labeled ABP GB123 provided the brightest signal while GB138
and GB137 produced similar signal accumulation to the
ProSense680 probe (Fig. 6A, B). Interestingly, although theProSense750 probe provided higher contrast in tumors at the early
time points compared to ProSense680, it produced substantially
weaker signal when compared to any of the other probes.
Finally, we removed tissues from mice after the 24 hr time point
and performed ex vivo imaging of the organs (Fig. 7A). This allowedus to confirm that the difference in tumor fluorescence observed in
the live animals was consistent with the signals observed in the
tumor tissues after removal. Furthermore, it allowed us to determine
the relative uptake of the probes into various major organs to
determine if there was a difference in background and probe
clearance (Fig. 7B). These data indicate that in all tissues other thanlung, the ABPs showed reduced uptake compared to the ProSense
substrates. Specifically we observed high signal in the kidney and
liver of ProSense treated mice.
Discussion
Recent advances in the development of new classes of optical
sensors have had a profound impact on the field of non-invasive
fluorescence imaging. While there have been significant improve-
ments in the instrumentation available for optical imaging
applications, even the most sensitive instruments cannot provide
molecular information that is critical for disease diagnosis and
detection using optical methods. Perhaps one of the most
promising new approaches for the development of optical contrast
agents is the development of ‘‘smart probes’’ that produce a
specific signal as the result of the action of a given enzymatic
target. The cysteine cathepsins have proven to be involved in the
regulation and mis-regulation of a number of key biological
processes associated with a wide range of diseases, including
cancer [15]. As a result, a number new methods have been
developed that take advantage of increased protease activity in and
around a diseased tissue of interest.
Several new classes of contrast agents have been designed to
target proteases in vivo. The majority of these agents are designedto act as substrates for a protease that, when hydrolyzed at a given
peptide bond, either relieve the quenching of a reporter, produce a
fluorescent byproduct or become retained in tissues at the site of
proteolysis [5,6,7,8]. As an alternative to substrate-based reporters,
we have recently described the use of small molecule activity based
probes that carry a fluorescent reporter [3,4]. These probes bind
to the active form of a protease and become permanently bound to
the active site nucleophile. ABPs can also be made in a quenched
form so that the fluorescent signal is only produced upon reaction
with the target protease [3]. The major difference between ABPs
and substrates is that the ABP binds to a single target protease and
inactivates it. Substrates, on the other hand, are processed and
turned over by the protease, leaving the enzyme active, thus
allowing for a potential amplification of signal as more substrate
molecules are processed. Because there had not been a careful
analysis of the benefits of signal amplification of substrates in vivo,we decided to carry out a study to directly compare commercially
available fluorescent substrates to our fluorescently labeled activity
based probes.
In this study we demonstrate in two different tumor types that
the large, polymer-based ProSense substrates are in fact less bright
than the small molecule ABPs. We believe that the reason why
amplification is not leading to a significantly brighter signal is that
these substrates are large molecules (.100,000 MW) that circulateslowly and diffuse slowly into tissues and cells. Once at the site of
proteolysis, they are processed to release small fluorescent
fragments that are then able to diffuse away from the site much
more readily. As a result, the amplification of signal at the site of
proteolysis does not have a dramatic enhancing effect for imaging.
Furthermore, the resulting fluorescent fragments are most likely
cleared through the liver and kidneys. This may explain why we
see significantly higher accumulation of fluorescent signal in the
kidney and liver of ProSense treated animals. Regardless of the
interpretation of these results, the data clearly demonstrate that
ABPs are able to produce signals that are at least as bright as the
signals observed for substrate probes.
As an alternative to large-polymer-based probes, there has also
recently been a report of small quenched peptide substrates that
were designed based on our ABP scaffold as well as on selective
inhibitor scaffolds in the patent literature [9]. These compounds
should display increased uptake and clearance similar to that
observed for our ABPs. We therefore anticipate that these reagents
will provide rapid activation, however, they are likely to suffer
from rapid clearance. In addition, all substrates suffer from the
significant drawback that they generally lack selectivity. Since the
only way to control selectivity is through the peptide recognition
sequence, it is often difficult to generate substrates that show
selective processing by a single protease or even family of
proteases. ABPs have the advantage of using highly specific
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Figure 4. Comparison of GB138 and ProSense680 using non-invasive optical imaging methods. (A) Images of GB138 and ProSense 680fluorescence in mice bearing MDA-MB 231 MFP tumors. Fluorescent images of live mice were taken at various time points after injection using anFMT1 imaging system. Representative pictures of the same mouse scanned over time in the 750 nm channel (GB138) and 680 nm channel(Prosense680) are shown. Fluorescent tomography scans (yellow box) are overlaid on an epifluoerscent image. The colorimetric scale bars indicatethe amount of fluorescence in the tomography box. (B) Quantification of total fluorescence in tumors. Fluorescence in tumors was measured in theindicated area (white box) and normalized to tumor weight. Mean fluorescence and standard error normalized to tumor weight for each channelswere plotted relative to time after probe injection. Insert shows tumor fluorescence (in pmol) normalized to weight at the 24 hour time point forGB138 and ProSense680 treated mice. GB138 fluorescence was corrected to account for the filter set of the FMT1 which is not optimal for the IR800fluorophore (see methods and Fig. S1). (C) Residual activity of cathepsins in tumors from GB138/ProSense680 treated mice. Residual cathepsin activitywas either directly labeled by addition of I125 JPM-OEt for 45 minutes, or samples were pretreated for 15 minutes with GB111-NH2 (a broad spectrumcathepsin inhibitor [3]) prior to I125 JPM-OEt labeling. Samples were analyzed by SDS-PAGE, followed by fluorescent scanning of the gel using anOdyssey scanner (left panel) followed by autoradiography (right panel).doi:10.1371/journal.pone.0006374.g004
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Figure 5. Comparison of all ABPs and substrate probes in parallel in separate mice. Fluorescence images of live mice bearing C2C12rastumors treated with GB137, GB123, GB138, ProSense750 and ProSense680. Images were taken at various time points after injection using theFMT2500 imaging system. Representative pictures of each mouse scanned over time in the 680 nm or 750 nM channel are presented. Fluorescenttomography scans in color are overlaid over an epifluorescent image in black and white. The colorimetric scale bars indicate nm of fluorescence.White circles indicate the location and rough size of individual tumors. These circles do not indicate the area used for quantification of tumorfluorescence in figure 6.doi:10.1371/journal.pone.0006374.g005
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functional group chemistry that dramatically restricts the potential
targets of the probes (i.e. only cysteine proteases will be covalently
modified by an AOMK probe). In addition, ABPs remain bound
to the target protease, leading to prolonged retention at the site of
interest (i.e. the tumor) and allowing subsequent assessment of
selectivity of target labeling using biochemical methods.
One of the potential drawbacks of using an ABP is the fact that
target binding leads to inhibition of enzymatic activity. To address
this issue, we have evaluated the extent of cysteine cathepsin
inhibition upon treatment of animals at the dose of the probe used
for imaging experiments. We show here that after treatment of
animals with the ABP probe GB138, we are able to see strong
residual labeling of target cathepsins by ex vivo addition of a
radiolabeled ABP. Thus, we find that there is virtually no
reduction in cysteine cathepsin protease activity compared to
mice treated with vehicle control. This suggests that, at the doses
used to generate optical imaging data, the probes are only
modifying and inhibiting a small percentage of the total pool of
active cathepsins. Therefore, we do not anticipate that probe
labeling will in any way impact disease progression or lead to toxic
effects as the result of inhibition of target proteases. Interestingly,
we found the ProSense signals were somewhat reduced when we
co-injected ABPs and substrates, suggesting that ABP inhibition of
target proteases may have reduced the ProSense signals to some
degree. This also suggests that the ProSense reagents are only
measuring a small percentage of active proteases, as their signal
can be reduced without inhibiting a significant portion of the total
active cathepsins.
Figure 6. Quantification of tumor fluorescence in mice treated in parallel with ABPs and ProSense substrates. (A) Plot of fluorescencein tumors from mice shown in Figure 5 at various time points after probe injection. The fluorescence in tumors was measured using a constant ROIand normalized to tumor weight. Mean fluorescence and standard error is plotted relative to time after probe injection. (B) Total tumor fluorescencein tumors excised 24 hours after probe injection and imaged ex vivo using the FMT2500 system. Mean fluorescence with standard error is shown.doi:10.1371/journal.pone.0006374.g006
Figure 7. Evaluation of probe distribution and tissue specificity. (A). Representative images of tissues from ProSense750 and GB138 treatedmice analyzed by ex vivo imaging using the FMT2500 imaging system. The relative scale for each set of tissues is indicated at the bottom. (B)Quantification of accumulation of probes in tumors relative to other major organs for each of the probes tested. The fluorescence measurementswere corrected for Cy5 and IR800 since the filter sets on the FMT system are not optimized for their maximum excitation/emission wavelengths (seemethods and Fig. S1).doi:10.1371/journal.pone.0006374.g007
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Taken together, our results suggest that both substrates and
ABPs are viable tools for imaging protease activity using non-
invasive imaging methods. However, our data suggest that the
assumption that substrates produce brighter signal as the result of
signal amplification is not valid, at least in the tumor models we
have analyzed here. We also demonstrate that, because of their
small size and overall rapid clearance in vivo, ABPs produce highcontrast images more rapidly and with lower background
retention in tissues such as liver and kidney compared to the
ProSense substrates. These properties coupled with the fact that
ABPs can be used to biochemically monitor protease targets after
imaging make them preferential to substrates for some imaging
applications. We are currently working to apply these and other
classes of fluorescent ABPs in additional mouse models of human
disease.
Materials and Methods
Fluorescent probesThe ProSense probes used in this study were purchased and
used as directed by the manufacturer (VisEn Imaging, Inc.). The
fluorescent ABPs were all synthesized and purified as described
previously [3,4].
Non-invasive imaging of tumor bearing miceAll animal experiments were approved by the Stanford
Administrative Panel on Animal Care and strictly followed their
specific guidelines. Male, 4–8 week-old nude mice (Nu/J 002019,
The Jackson Laboratory, Bar Harbor, Maine) were injected
subcutaneously with MDA-MB 231 MFP or C2C12ras cells
(1.56106). Tumors that were 30–100 mg in size formed 9 days to4 weeks after grafting. Mice were fed low fluorescent chow 3 days
prior to probe administration. Mice were injected either
individually with a single activity based probe or ProSense probe
or simultaneously with a mixture of ABP and ProSense probe
labeled with different wavelength fluorophores. A constant amount
of GB probes (25 nmols) and ProSense probes (2 nmol) was
administered by tail vein injection (e.g. 1.2 mg/ml GB123, 2 mg/
ml GB137, 1.6 mg/ml GB138 or 32 mg/ml ProSense 750 or
ProSense 680). All probes were injected in solution of 67% DMSO
33% PBS in 100 ml total volume. Prior to imaging, mice wereanesthetized with 3% isoflurane, starting prior to probe injection
and up to 24 hr after injection. Tomographic images of mice were
acquired using the FMT1 or FMT2500 imaging system using the
680 nm or 750 nm channels. Images are displayed as fluorescent
tomography scans in color overlaid on an epifluorescent image in
gray. The gain setting is kept constant for each probe throughout
the scanning series. After the last imaging point, mice were
humanely sacrificed and various organs were excised. Organs were
then imaged ex vivo using the FMT2500. To quantify non-invasiveimages, total fluorescence (in pmol) was recorded in identically
sized, 3 dimensional ROIs and normalized to tumor weight.
Fluorescence measurements of Cy5 containing probes e.g. GB123,
and GB137 were corrected to account for the use of a non-optimal
filter of ex/em of 670/700 nm (the 680 channel) rather then the
optimal 646/666 nm (Cy5 max Ex/Em). A correction factor was
determined by measuring the ratio of the predicted fluorescence of
a 100 pmol standard of GB123 with the 680 nm channel to the
actual measurement of the standard (a predicted 100 pmols was
measured as 16.3 pmol thus the scale factor was 6.12; Supple-
mental figure 1). Similarly, analysis of GB138 resulted in a scale
factor of 2.84 for this fluorophore. The ex vivo organ fluorescence
was measured using identical ROIs for each organ. Each ROI was
smaller than the organs in size, and the maximum signal in each
organ was recorded. Fluorescence of organs were normalized to
the area of the ROI.
Gel analysis of labeled tumorsTumors from mice injected with probes were excised and flash
frozen in liquid nitrogen. Tumors were lysed by dounce
homogenization or by bead beating of tissue in cold buffer (1%
Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate in PBS). For
radio-labeling studies 90 mg of protein in 30 ml of acetate buffer(50 mM sodium acetate pH 5.5, 5 mM MgCl2, and 4 mM DTT)
was treated with 2 ml I125-JPM-OEt (4.66106 counts min21 CPM)for 45 minutes. In addition, one control sample was pre-incubated
for 15 minutes with GB111-NH2 (a cathepsin inhibitor)[4] prior to
I125-JPM-OEt labeling. The reaction was stopped by addition of
sample buffer (10% glycerol, 50 mM Tris HCl, pH 6.8, 3% SDS
and 5% b-mercaptoethanol), boiled, and separated on a 12.5%
SDS-PAGE gel. Wet gels were first scanned for fluorescence using
an Odyssey scanner (LiCor Biosciences Nebraska USA) at 780/
800 nm and then dried and exposed to a phosphor imager plate
overnight. The plate was scanned with a Typhoon scanner.
Supporting Information
Figure S1
Found at: doi:10.1371/journal.pone.0006374.s001 (0.39 MB
PDF)
Acknowledgments
We would like to thank Elias Gounaris and Khashayarsha Khazaie
(Northwestern University) for helpful suggestions and critical evaluation of
the manuscript.
Author Contributions
Conceived and designed the experiments: GB MB. Performed the
experiments: GB RMW LEE WA. Analyzed the data: GB RMW LEE.
Wrote the paper: GB MB.
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